Abstract EANA2025-94

The exploration of life in extreme environments has highlighted cyanobacteria of the genus Chroococcidiopsis as prime candidates for astrobiology studies due to their remarkable resistance to solar and cosmic radiation, as evidenced by previous experiments performed outside the ISS and through ground-based irradiation campaigns. Notably, three isolates of Chroococcidiopsis, CCMEE 057, 064 and 029 in particular, exposed in both hydrated and dried state to different types of ionizing radiation (e.g., X-rays, Fe-ions) and UV-C radiation, showed outstanding radioresistance comparable to D. radiodurans. A key molecular feature contributing to such resilience may lie in specialized stress response proteins, particularly those involved in DNA protection and oxidative stress mitigation. Among them, Dps (DNA-binding proteins from starved cells) proteins are known to play a pivotal role in preventing DNA damage by physically shielding and scavenging reactive oxygen species through their ferroxidase activity. The final goal of this project is the characterization of Dps proteins and to identify novel proteins involved in radioresistance mechanisms in radiation-tolerant cyanobacteria, which may suggest new strategies to mitigate the radiation susceptibility of organisms relevant for developing biological systems to support human space exploration. Using an integrated experimental and computational approach, our aim is to characterize Dps activity and to assess their protective function when expressed in radiation-sensitive bacterial strains, in order to determine whether they confer enhanced DNA protection compared to non-transformed cells. Through in silico analysis, we identified four Dps proteins in CCMEE 029 by using the Dps sequences from the cyanobacterium Nostoc punctiforme to search for orthologs within the genome of CCMEE 029. Based on this, genes encoding the four Dps will be cloned into a shuttle plasmid capable of replicating in both E. coli and the cyanobacterium Synechocystis sp. PCC 6803, two radiosensitive model organisms; transformants will be exposed to increasing doses of gamma radiation (100 Gy to 1 kGy) to assess their radioresistance. Additionally, Chroococcidiopsis isolates CCMEE 029, 057, and 064 will be irradiated in the hydrated state with 5 kGy of gamma rays, and gene expression will be monitored at 3-, 6-, and 12-hours post-recovery. Furthermore, at least fifty Chroococcidiopsis isolates from the CCMEE will be exposed in the hydrated state to 5 kGy gamma radiation to identify novel genetic traits associated with radiation resistance through transcriptomics. In parallel, we aim to investigate the structural behaviour and DNA-binding affinity of the four CCMEE 029 Dps proteins through a computational approach, employing structural modeling and molecular dynamics simulations. The models of the functional dodecameric Dps and the supercoiled DNA structures were generated using AlphaFold 3 and NAB, respectively, and then manually assembled into DNA-Dps complexes using molecular visualization software. System preparation was carried out at different pH values, allowing assessment of DNA-binding activity under different pH conditions, and MD simulations were performed using AMBER. Binding energy calculations on the resulting MD trajectories will be used to compare the DNA-binding affinities of the four CCMEE 029 Dps proteins, to identify the one exhibiting the highest affinity and, consequently, the greatest potential for DNA protection.